Microbial Biotechnology: General Aspects PDF

Dr. Ilaria Benucci [email protected] Microbial Biotechnology General aspects Dr. Ilaria Benucci [email protected] INDEX 1. Microbial Biotechnology - Historical background 2. Biotechnology products...

Dr. Ilaria Benucci [email protected] Microbial Biotechnology General aspects Dr. Ilaria Benucci [email protected] INDEX 1. Microbial Biotechnology - Historical background 2. Biotechnology products 2.1 Biotechnology products - Microbial biomasses 2.2 Biotechnology products - Primary metabolites 2.3 Biotechnology products - Secondary metabolites Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background The biotechnological use of microorganisms has very ancient roots, in particular for the so-called classical or traditional biotechnologies which are based on the metabolism of one or more microorganisms for the transformation of many raw food materials. The identification, selection, cultivation and conservation of the microorganisms (starters) responsible of such processes is at the origin of modern biotechnology, guided and controlled transformation processes that have had a significant development over the last two centuries. Video: https://www.youtube.com/watch?v=wDun3LLOKL4&t Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background A clay tablet dating back to the Sumerian era (about 4,000 BC), better known as the "Blau monument" (from the archaeologist who discovered it) is the oldest evidence confirming the Sumerians' ability to brew beer. The tablet shows the propitiatory gifts offered to the goddess of fertility, including honey and, of course, beer. Barley and an ancient variety of spelt were ground to make a kind of loaf which was then soaked in water. Subsequently, the water with the cereals was heated and then, after cooling, the liquid was filtered. This "ancient" must was fermented by adding honey, to increase the fermentable sugars, and spices depending on the beer produced. Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background From traditional To modern biotechnology biotechnology In 1860 Louis Pasteur proved that the In 1883 is Emil production of wine, beer and other Christian Hansen fermentation processes are of microbial introduced the and not chemical origin. use of pure He built the basement of modern yeast cultures biotechnology Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background In 1860, Pasteur proved that the production of wine, beer and other fermentation processes are of microbial and not chemical origin. He then demonstrated that yeast can live with or without oxygen, multiplying in the first case, causing fermentation in the second. Only at the end of the nineteenth century we have gone from processes carried out in non-isolated systems (non-sterile), to processes carried out under controlled conditions (in sterility). Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background In 1883, for the first time Emil Christian Hansen https://www.chr- hansen.com/en/food-cultures-and- enzymes used a pure yeast culture to fine- tune the brewing process, at Carlsberg in Denmark, marking a significant advancement with respect to a very ancient process, dating back to the Sumerians. Pure culture was based on two principles: PURE CULTURE 1. bacteriological purity during the initial sowing; 2. the same purity during the production process. The wood of the tubs was then replaced by enamelled metal, then by copper in the 1920s and finally by stainless steel. The latter has been used since the 1960s. Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background The first industrial production in the history of yeast dates back to 1867, in Austria in the Mautner factory, where a baker's yeast was produced. The process consisted in preparing a grain mash in such a way that the development of carbon dioxide would bring the yeast to the surface where it was collected. After filtering, the yeast was first washed in cold water and collected in a large tank before being dried in screw presses or filter presses. In 1886, continuous ventilation of the culture environment was developed in England. The decisive advances occurred in Denmark and Germany between 1910 and 1920 with the process of progressive sugar feeding in the presence of oxygen. Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background In 1872 Baron Max de Springer of Vienna founded the first grain yeast factory in France, followed by Lesaffre, https://www.lesaffre.com/ and Bonduelle https://www.bonduelle.com/en/group /history.html. The yeast produced with the Viennese process quickly prevailed on the French market. This manufacturing process was used until the First World War. Dr. Ilaria Benucci [email protected] 1. Microbial Biotechnology Historical background Starting from the Second World War, biotechnological approaches have been the protagonists of the development of industrial production processes of penicillin and other antibiotics, while since the 1980s the commercial interest of fermentation processes has shifted towards small volumes of products with a high added value, in particular some molecules with biological activity to be used for the prevention or treatment of various pathologies. Furthermore, thanks to genetic techniques it has been possible to modify microorganisms in order to obtain molecules normally produced by animal cells, such as insulin, growth hormone, interferon and so on. The applications of these technologies have had an important impact on the fermentation industry, representing a decisive qualitative change, since it would not have been possible to achieve these results by using traditional techniques. Dr. Ilaria Benucci [email protected] HIGHLIGHTS 1. Microbial Biotechnology - Historical background - The biotechnological use of microorganisms has very ancient origin and dates back to Sumerian era. - In 1860 Pasteur proved that yeast can live with or without oxygen, multiplying in the first case, fermenting in the second. - In 1883 for the first time Emil Christian Hansen used a pure yeast culture to fine-tune the brewing process. - In 1867, in Austria the first industrial production of a baker's yeast was made. - In 1872 the first grain yeast factory was founded in France. Dr. Ilaria Benucci [email protected] 2. Biotechnology products The deliberate and controlled technological application of simple biological agents, such as microorganisms or their components, makes it possible to obtain goods and services, thanks to the integration of technological and scientific disciplines. The wide heterogeneity of microbial metabolism makes it possible to obtain a wide range of products. By exploiting the anaerobic metabolism it is possible to obtain products accumulated spontaneously in the microbial cells, such as lactic, propionic and butyric acids. Instead, the products of aerobic metabolism occupy an important position in the market of products obtainable from microorganisms. Very different products belong to this class, may be obtained both from primary microbial metabolism and from secondary biosynthetic metabolism. Cellular biomass Diagnostics Chemical comodities Valorization of residues Enzymes Treatment of solid and Specialties Goods Microorganisms Services liquid waste Dr. Ilaria Benucci [email protected] 2. Biotechnology products The products of primary metabolism (essential for the cell life and reproduction) can be divided into: cells and their constituents - microbial biomass, metabolic intermediates, which can be produced in excess with respect to physiological needs and accumulated inside or outside the cell. The products of secondary metabolism, which are not essential for cell development and reproduction, are generally accumulated outside the cell and do not always have a clear metabolic significance or function at the cellular level. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses The most easily obtainable class of products is microbial biomasses: ✓ mainly used for animal feed or as dietary supplements for humans, due to their content in proteins, vitamins and mineral salts. ✓ are obtained starting from substrates such as agro-industrial by- products or agricultural surplus. ✓ can be a valid alternative to conventional protein sources, as they are characterized by a protein content on dry matter of 50 - 80% in bacteria, 45 - 60% in yeasts, 30 - 55% in mold and 45 - 60% in algae. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses ✓ for animal feed - examples of biomasses achievable by microbiological processes are represented by Candida utilis (yeast) from sugary substrates, Methylococcus capsulatus (bacterium) from natural gas, Paecilomyces variotii (mold) from sulphite lye (residue from wood processing). ✓ for human nutrition, - among the microbial biomasses used we find Saccharomyces cerevisiae, which can be used as a supplement due to its content in mineral salts and B vitamins, mushrooms with fruiting bodies and the biomass obtainable by the QuornTM process. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses ✓ By the extraction from microbial biomass, different metabolites can also be obtained, such as cellular lysates (eg protein-vitamins), enzymes, lipids, nucleic acids and their derivatives, which can be used in the food and pharmaceutical sector. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses ✓ In the QuornTM process (https://www.quorn.co.uk/ ) the production of a fungal biomass for human nutrition from a strain of Fusarium venenatum was developed, and it may be used as a protein source alternative to meat. The mycoprotein obtained from the mushroom is collected, fermented, mixed with other nutrients (from wheat, corn,...) and then added with egg white or potato extract (vegan line). The different types of products (sausages, minced meat, meatballs, hamburgers, fillets, cubes, etc.) are steamed and then frozen. Video: https://www.youtube.com/watch?v=3wlprJOfNDA Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses Continuous fermentation in aerated bioreactors under axenic conditions (pure culture). Substrates composed of simple sugars (mainly pre-treated vegetable biomasses composed of more than 95% glucose), small sources of nitrogen and mineral salts (potassium, magnesium and phosphates). The cultivation takes place maintaining a pH equal to 6 and a temperature between 28 and 30°C; https://www.microbiologiaitalia.it/batteriologia/micoproteine-dalla-microbiologia-la-carne- sostenibile-del-futuro/ The substrate is continuously fed to the culture and when the cell biomass reaches a density of 25-30% w/v it is collected by filtration The biomass is then heat treated (64°C for at least 20 minutes) to reduce the ribonucleic acid content to levels permitted for human consumption. Too much ribonucleic acid can cause uric acid levels in the blood to rise. The heat treatment renders the insoluble hyphae with a malleable pasty consistency ideal for the production of meat-like preparations. the biomass is finally centrifuged and recovered. The product obtained is a semi-solid mass with a water content of 75% subsequently processed to obtain finished food products ready for use. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses ✓ The development of the production process of mushrooms with fruiting bodies (commonly known as mushrooms), started from the end of the 17th century in France. It allows us to obtain products such as Agaricus bisporus, including the hortensis variety (commercially known as champignon), Pleurotus ostreatus (orecchietta), Lentinula edodes (known as shiitake - culinary mushroom but also with medicinal properties rich in Vit. D) ✓ For other symbiotic genera, such as the species of Boletus (including the porcino) and Tuber (underground fungi, better known as truffles), natural processes are reproduced by infection protocols using the fungal spores of the roots of specific plants then planted. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Microbial biomasses ✓ Microbial biomass can also be produced and applied in vital form (starter cultures) to carry out their activity in complex medium, such as food matrices, or in biotransformation of specific substrates. ✓ One of the main applications is the production of Saccharomyces cerevisiae, a yeast used in wine, brewing and bread-making sector for its peculiar fermentation capabilities. Video: https://youtu.be/oTK9JNyCDN0 Video: https://www.britannica.com/video/185627/role-spoilage-cuisine-development 0.4 million metric tonnes of yeast biomass, including 0.2 million tonnes baker's yeast alone, are produced each year worldwide. Dr. Ilaria Benucci [email protected] HIGHLIGHTS 2.1 Biotechnology products - Microbial biomasses - For animal feed - For human nutrition (vegan food, cellular lysates, culinary mushroom ) - As starter cultures (to carry out their activity in complex medium, such as food matrices) Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter IndigenousWild Bread, wine, sake and beer are made with the Pure Yeast starter culture essential contribution of yeasts (mainly S. cerevisiae). As industrialization increased the manufacture Ancient practices were based on the natural of fermented products, the demand of yeast presence of this unicellular eukaryote, which grew exponentially. At the end of the 19th spontaneously starts the fermentation of century, addition of exogenous yeast sugars. biomass to produce bread and beer started to become a common practice. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Beet or cane molasses are the main substrate used in yeast production plants  Economically interesting (waste product coming ☓ Poor in some essential elements for yeast growth (nitrogen less from sugar refineries) than 3%; magnesium and phosphate)  sugars, mainly sucrose Contain 65-75% of ☓ Low content in vitamins (biotin, thiamine and pantothenic acid), required for fast growth. Must be  hydrolysed Sucrose is extracellularly supplemented by yeast in ☓ glucose and fructose Presence of different toxics that can affect yeast growth (herbicides, insecticides, ungicides, fertilizers and heavy metals) Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Pasteur effect (discovered in 1857 by Louis Pasteur) In aerobiosis, some yeasts including S. cerevisiae show a decrease in the speed of sugar consumption with partial and reversible inhibition of fermentation processes. If the culture is supplied with a sufficient quantity of oxygen, the yeast abandons its fermentative Yeast energy metabolism. Yeasts have two pathways for ATP production from metabolism, multiplying very actively. glucose, respiration, and fermentation. Both pathways start with glycolysis, which results in the production of two molecules of pyruvate and ATP per glucose. In It is known that respiration processes are more fermentation, pyruvate is then turned into ethanol. This process does not produce additional ATP but recycles the NAD+ consumed in glycolysis and thereby provides a favorable from a metabolic point of view (about 30 way of oxygen-independent ATP production. In respiration, pyruvate is completely oxidized to CO2 through the TCA cycle and oxidative phosphorylation (OXPHOS), moles of ATP per mole of glucose) than fermentation which yields additional ATP but requires oxygen. Crabtree positive yeasts, at sufficient levels of oxygen and glucose, use fermentation and respiration processes (2 moles of ATP per mole of glucose); simultaneously. The ethanol that accumulates in the environment can be recycled for ATP production once glucose has been depleted. This process, however, yields less therefore to reach the same cellular yield it is necessary ATP than the direct oxidation of pyruvate because the synthesis of Acetyl-CoA from ethanol requires ATP (Pfeiffer and Morley, 2014). a lower consumption of sugar in respiration. Consequently the glycolytic flux is lower in aerobiosis. Crabtree effect (or glucose repression) It is the phenomenon of a significant or total decrease in oxygen consumption by facultative fermenting microorganisms due to the presence of high glucose concentrations. This kind of microorganisms, in the presence of an excessive quantity of glucose (S. cerevisiae> 50 g / L), repress their aerobic oxidative processes (catabolic repression of the Krebs cycle). Video: https://www.exploreyeast.com/article/the-products-of-fermentation-yeast-video Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Diagram of the different stages in the industrial yeast BATCH biomass propagation process The first stage (F1) is initiated with a flask culture containing molasses, which is inoculated with the selected yeast strain. In a sequence of consecutive fermentations, the yeast biomass grown in small fermentors is used to inoculate larger tanks. In the initial batch phase (F2) , cells are exposed to BATCH FED-BATCH the high sugars concentration present in molasses. All the other nutrients are also present in the fermentor, and pH must be adjusted to 4.5-5.0. The presence of O2 from the beginning of the process allows yeast cells to synthesise lipids, thereby revitalising the sterol-deficient cell population and ensuring that fermentation can proceed efficiently Recent Advances in Yeast Biomass Production By Rocío Gómez-Pastor, Roberto Pérez-Torrado, Elena Garre and Emilia Matallana Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Diagram of the different stages in the industrial yeast BATCH biomass propagation process During batch fermentation (F2-F4) , a growth lag phase takes place in which cells synthesise the enzymes for the subsequent exponential phase. When the sugar concentration drops below a strain- specific level or the specific growth rate in aerobic cultures exceeds a critical value, a mixed respiro- BATCH FED-BATCH fermentative metabolism occurs (Crabtree effect). Alcoholic fermentation leads to a suboptimal biomass concentration but improve capacity by accumulating several necessary reserve metabolites to be used in the fed-batch phase. The presence of O2 during the process also allows yeast to oxidise alcoholic fermentation produced ethanol when sucrose is exhausted, which triggers the metabolism to change from fermentation to respiration, and eliminates ethanol from the media. Recent Advances in Yeast Biomass Production By Rocío Gómez-Pastor, Roberto Pérez-Torrado, Elena Garre and Emilia Matallana Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Diagram of the different stages in the industrial yeast From BATCH to FED-BATCH biomass propagation process As mentioned earlier, an oxygen supply is necessary to generate yeast biomass and to ensure optimal physiological conditions for effective fermentation. Oxygen is required for lipid synthesis, which is necessary to maintain plasma membrane integrity BATCH FED-BATCH and function, and consequently for both cell replication and the biosynthesis of sterols and unsaturated fatty acids. Recent Advances in Yeast Biomass Production By Rocío Gómez-Pastor, Roberto Pérez-Torrado, Elena Garre and Emilia Matallana Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Diagram of the different stages in the industrial yeast FED -BATCH biomass propagation process When ethanol is exhausted, the fed-batch phase starts (F5-F6). Optimisation of biomass productivity requires an increase in both the specific growth rate and the biomass yield during the fed-batch phase to the highest values possible under sugar-limited BATCH FED-BATCH cultivation. The growth rate profile during fed-batch cultivation is controlled primarily by the carbohydrate feedstock feed rate. The control of optimum dissolved oxygen during the fed-batch phase is also essential to obtain a high biomass yield. The pH and temperature are important parameters to be controlled during this phase: maintaining pH constantly at around 4.5 and maintaining temperature at 30ºC. Recent Advances in Yeast Biomass Production By Rocío Gómez-Pastor, Roberto Pérez-Torrado, Elena Garre and Emilia Matallana Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Video: https://www.youtube.com/watch?v=PDHb91wIGyg Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Video: https://www.youtube.com/watch?v=HHVOu3FT2oo Dr. Ilaria Benucci Oenological Properties Description [email protected] Fermenting power Maximum ethanol content (%, v/v) produced at the end of fermentation.  = Desirable  High alcohol-generating power. Fermenting vigor Amount of CO2 (g) produced in 24 hours of fermentation.  Fermentation starts quickly. ☓ = Not Desirable Selected strains should not release more than 100-400 mg/L Volatile acidity, Acetic acid production ☓ High production of volatile acidity/Acetic acid. Dispersed or flocculent growth, sedimentation rate. Growth mode in liquid medium  Dispersed growth during fermentation but quick sedimentation at the end of fermentation. Height of produced foam during fermentation. Foam formation ☓ High foam production. Thermotolerance/cryotolerance. Optimal fermentation temperature Optimal fermentation temperature 18-28 °C Antioxidant and antimicrobial agent.  High fermenting vigor and fermentation rate in presence of SO2 concentrations SO2 tolerance and production commonly used in cellar. Selection criteria for ☓ Excessive SO2 production. oenological S. The ability to degrade malic acid depends on the strain of S. cerevisiae, varying between 0 and 20% cerevisiae yeast strains Production or degradation of malic acid  Production/degradation of malic acid (It depends on the characteristics of the (source: Microbiologia must). Main secondary fermentation product contributing to sweetness, viscosity and enologica, Giovanna Suzzi e Glycerol production softness. Rosanna Tofalo)  Glycerol production range: 5-8 g/L Desired metabolite in Sherry, dessert wines and Porto. Essential factor for the Acetaldehyde production strains selection to be used in the aging of wines. Esters, higher alcohols, and volatile Moderate amounts of these compounds significantly affect the organoleptic compounds wines quality; their formation depends on the presence of precursors in musts. Off-flavour compound penalizing the wines organoleptic quality. H2S production Threshold values: 50-80 mg/L Nitrogenous compounds produced by microbial decarboxylation of amino acids. Few of these may cause food poisoning, due to their psychoactive and/or Biogenic amines production vasoactive properties.  Low producing strains. It is genotoxic and multipotent carcinogen in animals and, probably, carcinogenic in humans. Precursors present in musts (e.g. hydrogen cyanide, urea and ethanol) Ethyl carbamate production can lead to the ethyl carbamate formation during fermentation.  Low potential for ethyl carbamate production. Stress resistance  Tolerance to osmotic stress. High copper concentrations in musts may cause stuck fermentation. Copper resistance  Resistance to copper and to be able to assimilate the copper. Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter A new production process of Active Dry Yeasts - YSEO® (from Yeast SEcurity Optimization) Researchers from Lallemand, at Washington State University (USA), have developed and validated a natural preparation of Active Dry Yeast (ADY) that increases the effectiveness and regularity of fermentation. This process, called YSEO® (from Yeast Security Optimization), is based on the optimization of the nutritional strategy of the yeast during the multiplication phase of the culture that precedes the drying of the yeasts. This nutritional strategy increases the effectiveness of ADY preparations and allows fermentation to be completed even under difficult conditions. Video: https://www.youtube.com/watch?v=KxlD6V5iFNk http://www.lallemandwine.com/wp-content/uploads/2014/07/Brochure-YSEO3.pdf Dr. Ilaria Benucci [email protected] 2.1 Biotechnology products Production of microbial biomasses - starter Dr. Ilaria Benucci [email protected] HIGHLIGHTS 2.1 Biotechnology products – Production of microbial biomasses - starter - Pasteur effect; - Crabtree effect; - Industrial production of microbial biomasses to be used as starter cultures. Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites ✓ Final or intermediate products of energy metabolism, mainly poured out of the cell, into the culture medium, from which they are extracted and then purified. ✓ Can result from anaerobic metabolism, as final products, for example lactic acid from Lactobacillus, propionic acid from Propionibacterium and butyric acid from Clostridium. ✓ Spontaneously accumulated, even at significant amounts Lactobacillus delbrüeckii, ssp. Bulgaricus can produce up to 130 - 140 g of L-lactic acid per liter of culture. Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Lactic Acid production by Lactic Acid Bacteria (LAB)  Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Polylactic acid (PLA) ✓ thermoplastic polymer belonging to the family of aliphatic polyesters, with properties similar to Polyester and PET; ✓ biodegradable and 100% compostable, transparent, produced starting from sugar cane or glucose; ✓ easily processable by using normal standard machines; ✓ it has excellent resistance to oils and fats and to numerous chemical agents. The preparation of Polylactic Acid (PLA) occurs in 2 distinct stages: synthesis by fermentation and isolation ✓ good barrier to aromas and oxygen, low barrier to of L-lactic acid, polymerization of the obtained acid. Separation of starch from fiber and gluten water vapor; Liquefaction and saccharification of starch Fermentation with reuse (in the culture broth) of the ✓ degrades, without leaving polluting elements, in protein part separated from the starch Purification and concentration of lactic acid salt 15 months if left on the ground, in 24 months if solutions Polymerization buried, 48 months in water. Preparation of the product Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Polylactic acid (PLA) PLA is conventionally applied for: ✓ Fiber extrusion: tea bags and clothing; ✓ Injection molding: jewelry boxes; ✓ Compound: with wood and PMMA; ✓ Thermoforming: bivalve containers, trays for sweets, cups and pods for coffee; ✓ Blow molding: water bottles (not added with gas), fresh juices and bottles for cosmetics. Video: https://www.youtube.com/watch?v=JgrevlhUSNI Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Extracellular polymers synthesized by some Lactic acid bacteria (LAB) According to the chemical composition and biosynthesis mechanisms, EPS are classified into two distinct groups: Homopolysaccharides (HoPS). Heteropolysaccharides (HePS). Video: https://www.youtube.com/watch?v=Mk27FgGbv1c Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Homopolysaccharides (HoPS) HoPS are mainly synthetised extracellularly from an existing sucrose molecule, which act as donor of the corresponding monosaccharide by action of extracellular enzyme belonging to the glycosyl hydrolase family using sucrose as the glycosyl (fructose or glucose) donor The key enzymes are: glucansucrases and fructansucrases HoPS are composed of a single type of monosaccharide, glucose or fructose. These α- or β-D-glucans and β-fructans are polymers with high molar mass (≥ 1 x 106 Da) and may have a different branching degree. They are usually produced in quantities greater than 1g/L, although this value varies depending on the production conditions. Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Heteropolysaccharides (HePS) HePS synthesis differs from HoPS synthesis since the precursor repeating units are formed intracellularly and isoprenoid glycosyl carried lipids are involved in the process. The repeating units are translocated across the membrane and subsequently polymerized extracellularly. HePS are composed of a backbone of repeated subunits that are branched or unbranched, and consist of three to eight monosaccharides, (D-glucose, D- galactose, and L-rhamnose) derivatives of monosaccharides (N-acetylglucosamine, N-acetylgalactosamine or glucuronic acid) or substituted monosaccharides (such as phosphate, acetyl, and glycerol). Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Source: Bajpai et al., 2021 Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Source: Zannini et al., 2016 Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB Source: Zannini et al., 2016 Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Exopolysaccharides (EPS) Production by LAB ✓ homopolysaccharides can be introduced into naturally leavened products because they improve the quality of the structure, the cooking capacity and reduce the refining factors of bread; ✓ heteropolysaccharides are widely used as additives in dairy products. An example for the industrial use of EPS in baked goods is the application of dextran in panettone and other types of bread. Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites Organic compounds ✓ citric acid from the aerobic metabolism, directly as intermediates of the TCA tricarboxylic acid cycle (or Krebs cycle). The production of citric acid (120 - 150 g of citric acid per liter of culture) may be obtained by Aspergillus niger using carbohydrate substrates, and by the yeast Yarrowia lipolytica using n-alkanes as substrates. ✓ Ethanol may be produced by the fermentation of substrates rich in sugars (mainly agro-industrial residues or agricultural surplus), mainly using Saccharomyces cerevisiae yeast. Dr. Ilaria Benucci [email protected] 2.2 Biotechnology products Primary metabolites ✓ amino acids are primary metabolites achievable Organic compounds from microorganisms, with procedures that are an alternative to the isolation from proteins. ✓ Glutamic acid is obtained using bacterial strains such as Corynebacterium glutamicum and Flavobacterium flavum, by the action of a specific dehydrogenase that acts on α-ketoglutaric acid (an intermediate of the TCA cycle). ✓ From these same bacterial strains it is possible to produce lysine, alternatively produced by Enterobacter aerogenes, by transformation of the diaminopimelic acid precursor, obtainable in turn from Escherichia coli. Dr. Ilaria Benucci [email protected] HIGHLIGHTS 2.2 Biotechnology products - Primary metabolites - Lactic acid from Lactobacillus, propionic acid from Propionibacterium and butyric acid from Clostridium - Polylactic acid (PLA). - Exopolysaccharides produced by LAB. - Organic compounds (citric acid, ethanol, amino acids) Dr. Ilaria Benucci [email protected] 2.3 Biotechnology products Secondary metabolites ✓ Products having biological activity such as antimicrobials, immunomodulators, antitumors, inhibitors of specific enzymatic activities and growth promoters. ✓ A significant example is represented by the antibiotics obtainable from bacterial and fungal strains. ✓ Among the other secondary metabolites with biological activity, current interest is for statins, Video: molecules with anticolesterolemic activity https://microbiologysociety.org/our- work/75th-showcasing-why- obtainable from fungal strains of Aspergillus microbiology-matters/microbes- and-where-to-find-them/novel- terreus and species of the genus Monascus. products-from-microbes.html  HIGHLIGHTS Dr. Ilaria Benucci [email protected] 2.3 Biotechnology products - Secondary metabolites - Products with biological activity such as antimicrobials, immunomodulators, antitumors, inhibitors of specific enzymatic activities, growth promoters and statins